Longitudinal tracking of neuronal mitochondria delineates PINK1/Parkin-dependent mechanisms of mitochondrial recycling and degradation.

Altered mitochondrial quality control and dynamics may contribute to neurodegenerative diseases, including Parkinson's disease, but we understand little about these processes in neurons. We combined time-lapse microscopy and correlative light and electron microscopy to track individual mitochondria in neurons lacking the fission-promoting protein dynamin-related protein 1 (Drp1) and delineate the kinetics of PINK1-dependent pathways of mitochondrial quality control. Depolarized mitochondria recruit Parkin to the outer mitochondrial membrane, triggering autophagosome formation, rapid lysosomal fusion, and Parkin redistribution. Unexpectedly, these mitolysosomes are dynamic and persist for hours. Some are engulfed by healthy mitochondria, and others are deacidified before bursting. In other cases, Parkin is directly recruited to the matrix of polarized mitochondria. Loss of PINK1 blocks Parkin recruitment, causes LC3 accumulation within mitochondria, and exacerbates Drp1KO toxicity to dopamine neurons. These results define a distinct neuronal mitochondrial life cycle, revealing potential mechanisms of mitochondrial recycling and signaling relevant to neurodegeneration.

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism.

Overexpression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protect against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations, and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, noncanonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70's canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same noncanonical mechanism.

Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions.

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.

Glu-C, an alternative digestive enzyme for the quantitative LC-MS/MS analysis of an IgG-based antibody biotherapeutic.

AIM: LC-MS/MS bottom-up quantitation of proteins has become increasingly popular with trypsin as the most commonly used protease. However, trypsin does not always yield suitable surrogate peptides. An alternative enzyme, Glu-C, was used to generate surrogate peptides for quantifying a bispecific IgG1 biotherapeutic antibody in preclinical matrices.  Materials and methods: IgG1 was quantified by pellet digestion using an Acquity UPLC coupled  with a Xevo TQ-S mass spectrometer.  Results: Two generic LC-MS/MS methods (heavy and light chain) were developed which afforded acceptable precision and accuracy, and an lower limit of quantitation of 1 µg/ml in three preclinical matrices. A small nonsignificant bias was observed when cynomolgus serum LC-MS/MS results were compared with electrochemiluminescent immunoassay data. CONCLUSION: Glu-C was successfully used as an alternative digestion enzyme for bottom-up quantitation of an IgG1 in matrices from multiple preclinical species, with good agreement with electrochemiluminescent immunoassay data.

Loss of α-Synuclein Does Not Affect Mitochondrial Bioenergetics in Rodent Neurons.

Increased α-synuclein (αsyn) and mitochondrial dysfunction play central roles in the pathogenesis of Parkinson's disease (PD), and lowering αsyn is under intensive investigation as a therapeutic strategy for PD. Increased αsyn levels disrupt mitochondria and impair respiration, while reduced αsyn protects against mitochondrial toxins, suggesting that interactions between αsyn and mitochondria influences the pathologic and physiologic functions of αsyn. However, we do not know if αsyn affects normal mitochondrial function or if lowering αsyn levels impacts bioenergetic function, especially at the nerve terminal where αsyn is enriched. To determine if αsyn is required for normal mitochondrial function in neurons, we comprehensively evaluated how lowering αsyn affects mitochondrial function. We found that αsyn knockout (KO) does not affect the respiration of cultured hippocampal neurons or cortical and dopaminergic synaptosomes, and that neither loss of αsyn nor all three (α, ß and γ) syn isoforms decreased mitochondria-derived ATP levels at the synapse. Similarly, neither αsyn KO nor knockdown altered the capacity of synaptic mitochondria to meet the energy requirements of synaptic vesicle cycling or influenced the localization of mitochondria to dopamine (DA) synapses in vivo. Finally, αsyn KO did not affect overall energy metabolism in mice assessed with a Comprehensive Lab Animal Monitoring System. These studies suggest either that αsyn has little or no significant physiological effect on mitochondrial bioenergetic function, or that any such functions are fully compensated for when lost. These results implicate that αsyn levels can be reduced in neurons without impairing (or improving) mitochondrial bioenergetics or distribution.

Loss of mitochondrial fission depletes axonal mitochondria in midbrain dopamine neurons.

Disruptions in mitochondrial dynamics may contribute to the selective degeneration of dopamine (DA) neurons in Parkinson's disease (PD). However, little is known about the normal functions of mitochondrial dynamics in these neurons, especially in axons where degeneration begins, and this makes it difficult to understand the disease process. To study one aspect of mitochondrial dynamics-mitochondrial fission-in mouse DA neurons, we deleted the central fission protein dynamin-related protein 1 (Drp1). Drp1 loss rapidly eliminates the DA terminals in the caudate-putamen and causes cell bodies in the midbrain to degenerate and lose α-synuclein. Without Drp1, mitochondrial mass dramatically decreases, especially in axons, where the mitochondrial movement becomes uncoordinated. However, in the ventral tegmental area (VTA), a subset of midbrain DA neurons characterized by small hyperpolarization-activated cation currents (Ih) is spared, despite near complete loss of their axonal mitochondria. Drp1 is thus critical for targeting mitochondria to the nerve terminal, and a disruption in mitochondrial fission can contribute to the preferential death of nigrostriatal DA neurons.

Energy failure: does it contribute to neurodegeneration?

Energy failure from mitochondrial dysfunction is proposed to be a central mechanism leading to neuronal death in a range of neurodegenerative diseases. However, energy failure has never been directly demonstrated in affected neurons in these diseases, nor has it been proved to produce degeneration in disease models. Therefore, despite considerable indirect evidence, it is not known whether energy failure truly occurs in susceptible neurons, and whether this failure is responsible for their death. This limited understanding results primarily from a lack of sensitivity and resolution of available tools and assays and the inherent limitations of in vitro model systems. Major advances in these methodologies and approaches should greatly enhance our understanding of the relationship between energy failure, neuronal dysfunction, and death, and help us to determine whether boosting bioenergetic function would be an effective therapeutic approach. Here we review the current evidence that energy failure occurs in and contributes to neurodegenerative disease, and consider new approaches that may allow us to better address this central issue.

A neo-substrate that amplifies catalytic activity of parkinson's-disease-related kinase PINK1.

Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.

PSD-95 expression controls L-DOPA dyskinesia through dopamine D1 receptor trafficking.

L-DOPA-induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson's disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson's patients.

L-DOPA impairs proteasome activity in parkinsonism through D1 dopamine receptor.

Aberrant membrane localization of dopamine D(1) receptor (D1R) is associated with L-DOPA-induced dyskinesia (LID), a major complication of L-DOPA treatment in Parkinson's disease (PD). Since the proteasome plays a central role in modulating neuronal response through regulation of neurotransmitter receptor intraneuronal fate, we hypothesized that the ubiquitine-proteasome proteolytic pathway could be impaired in LID. Those LIDs are actually associated with a striatum-specific decrease in proteasome catalytic activity and accumulation of polyubiquitinated proteins in experimental rodent and monkey parkinsonism. We then demonstrated that such decreased proteasome catalytic activity (1) results from D1R activation and (2) feed-back the D1R abnormal trafficking, i.e., its exaggerated cell surface abundance. We further showed that the genetic invalidation of the E3 ubiquitin-protein ligase parkin PD gene leads to exaggerated abnormal involuntary movements compared with wild-type mice. We thus established in an unprecedented series of experimental models that impairment of the ubiquitine-proteasome system at specific nodes (E3 ligase parkin, polyubiquitination, proteasome catalytic activity) leads to the same phenomenon, i.e., aberrant behavioral response to dopamine replacement therapy in PD, highlighting the intimate interplay between dopamine receptor and proteasome activity in a nondegenerative context.

Endogenous morphine-like compound immunoreactivity increases in parkinsonism.

Morphine is endogenously synthesized in the central nervous system and endogenous dopamine is thought to be necessary for endogenous morphine formation. As Parkinson's disease results from the loss of dopamine and is associated with central pain, we considered how endogenous morphine is regulated in the untreated and l-DOPA-treated parkinsonian brain. However, as the cellular origin and overall distribution of endogenous morphine remains obscure in the pathological adult brain, we first characterized the distribution of endogenous morphine-like compound immunoreactive cells in the rat striatum. We then studied changes in the endogenous morphine-like compound immunoreactivity of medium spiny neurons in normal, Parkinson's disease-like and l-DOPA-treated Parkinson's disease-like conditions in experimental (rat and monkey) and human Parkinson's disease. Our results reveal an unexpected dramatic upregulation of neuronal endogenous morphine-like compound immunoreactivity and levels in experimental and human Parkinson's disease, only partially normalized by l-DOPA treatment. Our data suggest that endogenous morphine formation is more complex than originally proposed and that the parkinsonian brain experiences a dramatic upregulation of endogenous morphine immunoreactivity. The functional consequences of such endogenous morphine upregulation are as yet unknown, but based upon the current knowledge of morphine signalling, we hypothesize that it is involved in fatigue, depression and pain symptoms experienced by patients with Parkinson's disease.

Double-dissociation of the catecholaminergic modulation of synaptic transmission in the oval bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BST) is a cluster of nuclei within the extended amygdala, a forebrain macrostructure with extensive projection to motor nuclei of the hindbrain. The subnuclei of the BST coordinate autonomic, neuroendocrine, and somato-motor functions and receive robust neuromodulatory monoaminergic afferents, including 5-HT-, noradrenaline (NA)-, and dopamine (DA)-containing terminals. In contrast to 5-HT and NA, little is known about how DA modulates neuronal activity or synaptic transmission in the BST. DA-containing afferents to the BST originate in the ventral tegmental area, the periaqueducal gray, and the retrorubral field. They form a fairly diffuse input to the dorsolateral BST with dense terminal fields in the oval (ovBST) and juxtacapsular (jxBST) nuclei. The efferent-afferent connectivity of the BST suggests that it may play a key role in motivated behaviors, consistent with recent evidence that the dorsolateral BST is a target for drugs of abuse. This study describes the effects of DA on synaptic transmission in the ovBST. Whole cell voltage clamp recordings were performed on ovBST neurons in brain slices from adult rats in the presence or absence of exogenous DA and receptor-targeted agonists and antagonists. The results showed that DA selectively and exclusively reduced inhibitory synaptic transmission in the ovBST in a dose-dependent and D2-like dopamine receptor-dependent manner. DA also modulated excitatory synaptic transmission in a dose-dependent dependent manner. However, this effect was mediated by α2-noradrenergic receptors. Thus these data reveal a double dissociation in catecholaminergic regulation of excitatory and inhibitory synaptic transmission in the ovBST and may shed light on the mechanisms involved in neuropathological behaviors such as stress-induced relapse to consumption of drugs of abuse.

Lentiviral overexpression of GRK6 alleviates L-dopa-induced dyskinesia in experimental Parkinson's disease.

Parkinson's disease is caused primarily by degeneration of brain dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine in the striatum. Dopamine replacement therapy with the dopamine precursor l-dopa is the mainstay of current treatment. After several years, however, the patients develop l-dopa-induced dyskinesia, or abnormal involuntary movements, thought to be due to excessive signaling via dopamine receptors. G protein-coupled receptor kinases (GRKs) control desensitization of dopamine receptors. We found that dyskinesia is attenuated by lentivirus-mediated overexpression of GRK6 in the striatum in rodent and primate models of Parkinson's disease. Conversely, reduction of GRK6 concentration by microRNA delivered with lentiviral vector exacerbated dyskinesia in parkinsonian rats. GRK6 suppressed dyskinesia in monkeys without compromising the antiparkinsonian effects of l-dopa and even prolonged the antiparkinsonian effect of a lower dose of l-dopa. Our finding that increased availability of GRK6 ameliorates dyskinesia and increases duration of the antiparkinsonian action of l-dopa suggests a promising approach for controlling both dyskinesia and motor fluctuations in Parkinson's disease.

Pharmacological analysis demonstrates dramatic alteration of D1 dopamine receptor neuronal distribution in the rat analog of L-DOPA-induced dyskinesia.

We have associated behavioral, pharmacological, and quantitative immunohistochemical study in a rat analog of l-DOPA-induced dyskinesia to understand whether alterations in dopamine receptor fate in striatal neurons may be involved in mechanisms leading to movement abnormalities. Detailed analysis at the ultrastructural level demonstrates specific alterations of dopamine D(1) receptor (D(1)R) subcellular localization in striatal medium spiny neurons in l-DOPA-treated 6-hydroxydopamine-lesioned rats with abnormal involuntary movements (AIMs). This includes exaggerated D(1)R expression at the plasma membrane. However, D(1)R retains ability of internalization, as a challenge with the potent D(1)R agonist SKF-82958 induces a strong decrease of labeling at membrane in animals with AIMs. Since a functional cross talk between D(1)R and D(3)R has been suggested, we hypothesized that their coactivation by dopamine derived from l-DOPA might anchor D(1)R at the membrane. Accordingly, cotreatment with l-DOPA and the D(3)R antagonist ST 198 restores normal level of membrane-bound D(1)R. Together, these results demonstrate that AIMs are related to abnormal D(1)R localization at the membrane and intraneuronal trafficking dysregulation, and suggest that strategies aiming at disrupting the D(1)R-D(3)R cross talk might reduce l-DOPA-induced dyskinesia by reducing D(1)R availability at the membrane.

Dopamine receptors and L-dopa-induced dyskinesia.

In the majority of Parkinson's disease patients, chronic dopamine replacement therapy leads to involuntary aimless movements known as l-dopa-induced dyskinesia. While mechanisms involved in dyskinesia occurrence are still unclear, dopamine receptors undoubtedly have a central role in their pathophysiology. Here we review current knowledge and evidence for their involvement in dyskinesia genesis and manifestation. We propose that an anti-dyskinetic strategy should target the D1/D3 signalling cascade, as targeting D2 receptor signalling seems to inherently convey anti-therapeutic effects deleterious to patients. As more molecular tools are made available, we will better understand the role of each receptor and its associated signalling cascade in Parkinson's disease and L-dopa-induced dyskinesia, hopefully in a way amenable to patients.

Antagonizing L-type Ca2+ channel reduces development of abnormal involuntary movement in the rat model of L-3,4-dihydroxyphenylalanine-induced dyskinesia.

BACKGROUND: Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment of Parkinson's disease (PD) leads to debilitating involuntary movements, termed L-DOPA-induced dyskinesia. Striatofugal medium spiny neurons (MSN) lose their dendritic spines and cortico-striatal glutamatergic synapses in PD and in experimental models of DA depletion. This loss of connectivity is triggered by a dysregulation of intraspine Cav1.3 L-type Ca2+ channels. Here we address the possible implication of DA denervation-induced spine pruning in the development of L-DOPA-induced dyskinesia. METHODS: The L-type Ca2+ antagonist, isradipine was subcutaneously delivered to rats at the doses of .05, .1, or .2 mg/kg/day, for 4 weeks, starting the day after a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion. Fourteen days later, L-DOPA treatment was initiated. RESULTS: Isradipine-treated animals displayed a dose-dependent reduction in L-DOPA-induced rotational behavior and abnormal involuntary movements. Dendritic spine counting at electron microscopy level showed that isradipine (.2 mg/kg/day) prevented the 6-OHDA-induced spine loss and normalized preproenkephalin-A messenger RNA expression. Involuntary movements were not reduced when isradipine treatment was started concomitantly with L-DOPA. CONCLUSIONS: These results indicate that isradipine, at a therapeutically relevant dose, might represent a treatment option for preventing L-DOPA-induced dyskinesia in PD.